Congenital thrombotic thrombocytopenic purpura (cTTP) is an ultra-rare, life-threatening hereditary disorder that causes patients to experience significant morbidity and decreased health-related quality of life (HRQoL). A cTTP disease-specific patient-reported outcome (PRO) instrument that is reflective of patients’ experiences with the disorder does not currently exist. The objective of this study was to evaluate and validate the psychometric properties of the Congenital Thrombotic Thrombocytopenic Purpura–Patient Experience Questionnaire (cTTP-PEQ), developed using a literature review, interviews with expert clinicians, and qualitative concept elicitation and cognitive debriefing interviews.This prospective, observational study (NCT03519672) was conducted with patients diagnosed with cTTP currently receiving treatment. Patients were enrolled through investigator sites and direct-to-patient recruitment. Individuals completed electronic self-administered PRO measures, including the cTTP-PEQ, at baseline and Day 14 (+ up to 10 days). The cTTP-PEQ consisted of five multi-item domains  and three single-item domains , and assessed symptoms and impact of cTTP in the previous 24 h, 7 days, and 2 weeks. Convergent and discriminant validity were evaluated using Spearman’s rank correlation coefficients. Known-groups validity was assessed between patient groups separated by Patient Global Impression of Severity . Internal reliability was assessed using Cronbach’s alpha. Test–retest reliability was assessed using intraclass correlation coefficients (ICCs).r ≥ 0.4) for 12/15 hypothesized relationships between pairs of domains and/or total scores. Discriminant validity was confirmed with low correlations (r < 0.3) observed for 5/7 hypothesized relationships. Known-groups validity was confirmed with significant differences (p ≤ 0.05) in mean cTTP-PEQ scores between the two PGI-S groups for most domains and items at both timepoints. Cronbach’s alpha was 0.88 at baseline and 0.91 at Day 14, confirming internal consistency of the instrument. Test–retest reliability was also confirmed with a high ICC (0.96).Thirty-six patients participated in this study. Convergent validity was confirmed with high-to-moderate correlations (This study validates the psychometric properties of the novel cTTP-PEQ for use in research and clinical practice to assess HRQoL among patients with cTTP. This instrument will be particularly useful when assessing cTTP disease burden and the impact of new treatments.The online version contains supplementary material available at 10.1186/s41687-023-00592-w. Thrombotic thrombocytopenic purpura (TTP) is a rare, life-threatening blood disorder resulting from a deficiency in the clotting enzyme ADAMTS13  . This leThe spectrum of severity of cTTP can be extremely varied, including asymptomatic disease, single episode and chronic-relapsing forms, and multiorgan failure . The claAlthough there is no specific therapy approved for TTP treatment, many patients require ongoing prophylactic treatment with intravenous plasma infusion, typically administered every 2 to 3 weeks to help alleviate symptoms and reduce long-term complications , 13. vonAccurate assessment of disease- and treatment-related burden is essential to improve our understanding of the effect of cTTP on patients’ HRQoL and to assess the impact of new treatments. Therefore, it is important that any patient-reported outcome (PRO) instrument measures the concepts most relevant to a patient’s specific disease condition and treatment. Several generic PRO instruments have been used to assess the HRQoL of patients with TTP. These include using the Medical Outcomes Study 36-Item Short-Form Health Survey (SF-36) to assess physical and cognitive long-term deficits in HRQoL after recovery , and theTherefore, a PRO instrument that was comprehensive and specific for cTTP, whilst having a minimal burden on the patient to complete, would be valuable for the clinical management of cTTP. A conceptual model capturing the symptoms and impacts of cTTP has previously been formulated . This moConcept elicitation and development of a cTTP-specific PRO instrument was conducted in accordance with good practice guidance for establishing content validity . The conA scoring algorithm for the cTTP-PEQ was developed, with most items scored using a numerical scale ranging from 0 to 4, 0 to 5, or 0 to 10 Table . FatigueThe cTTP-PEQ was evaluated in a prospective, non-interventional, observational cohort study (NCT03519672) conducted at three study sites that treat patients with rare hematologic conditions between July 2018 and April 2019. Two of the study sites were in the United Kingdom  and one was in the United States . Owing to the inherent difficulties associated with recruiting a representative sample of patients with cTTP, patients were also enrolled by direct-to-patient recruitment using two third-party qualified vendors that specialize in recruiting patients with rare diseases, facilitated by a contract research organization. Adult (≥ 18 years of age) and adolescent (12–17 years of age) patients with the ability to read, write, and speak English were eligible for inclusion. Eligible patients had a diagnosis of cTTP and were currently receiving prophylactic or on-demand treatment with fresh frozen plasma, solvent detergent plasma, or VWF/FVIII concentrate. Patients participating in a concurrent clinical trial were excluded from the study. All patients provided informed consent for participation, and were screened and followed up using the same process, regardless of recruitment method. For patients recruited using the direct-to-patient approach, verification of the cTTP diagnosis was provided by the patient’s treating physician.Following recruitment, information on patients’ sociodemographic characteristics and cTTP-related clinical measures, including disease severity, presence and number of comorbidities, and cTTP treatment history, were recorded. Using either the study site or their own internet-connected computer or device, each patient completed eight self-administered, electronically formatted PRO measures, including the cTTP-PEQ. As patients receiving prophylactic plasma infusions typically undergo treatment every 2 to 3 weeks, a repeat testing interval of 14 days (+ up to 10 days) from baseline was selected. Patients completed the same set of electronically formatted PRO measures at Day 14 (+ 10 days) following their first assessment. Changes in patient health status from baseline were also assessed using the Patient Global Impression of Change (PGI-C) scale .In addition to the cTTP-PEQ, patients completed eight existing PRO measures to assess the psychometric properties of the newly developed cTTP-specific instrument. These included the Patient-Reported Outcomes Measurement Information System (PROMIS®)-29 Profile , the HITQuantitative data from patient characteristics and each individual PRO measure were summarized using descriptive statistics, which were calculated for each item, subscale or domain score, and total score, when applicable, at baseline and Day 14. Due to the non-interventional nature of the study, missing values were expected for the second assessment owing to patient drop-out. Patients who did not complete the second assessment, despite multiple reminders, were assumed missing at random. Missing and invalid observations were recorded as a separate category, and missing data were not imputed. Percentages of patients were based on the number of patients who completed all items. Analyses were based on the available data for item-specific analyses and on observations with full data for multi-item domains and totals or multiple timepoints.To assess the strength of hypothesized relationships between the cTTP-PEQ and the existing PRO measures, Spearman’s rank correlation coefficients were used to evaluate convergent and discriminant validity at baseline and Day 14. The hypothesized relationships between the cTTP-PEQ domains and existing PRO measures expected to demonstrate “moderate-to-high” or “low” correlation are detailed in Table S2. Correlation coefficient values ≤ 0.19 were considered “very low,” values between 0.20 and 0.29 were considered “low,” values between 0.30 and 0.49 were considered “moderate,” and those between 0.50 and 0.69 were considered “high.” Correlation coefficients ≥ 0.70 were considered “very high” .Known-groups validity was assessed at baseline and on Day 14 by comparing the mean cTTP-PEQ scores between patient groups separated by PGI-S score . Significance was defined as an alpha value ≤ 0.05.Item-to-item and item-to-total correlations, estimated using Spearman’s rank correlation coefficients, were used to support the grouping of items measuring a similar construct in order to assess the internal reliability of the conceptual model. Internal consistency was assessed using Cronbach’s alpha, which was calculated for the total cTTP-PEQ score and for all subscales separately. Values of 0.70–0.95 were considered adequate for group-level comparisons. Test–retest reliability was assessed for each domain and total score of the cTTP-PEQ by calculating the intraclass correlation coefficient (ICC) between baseline and Day 14 for patients who reported no change in PGI-C score. Values of 0.4–0.75 were considered to represent fair-to-good reliability and values > 0.75 represent excellent reliability.A total of 38 patients were enrolled in the study. Two of these patients were adolescents and 36 were adults. Owing to the small sample size of the adolescent population, these patients were excluded from the analysis, leaving a total of 36 adult patients in the full analysis population. Baseline characteristics are shown in Table Descriptive statistics for the cTTP-PEQ item and domain scores at baseline and Day 14 are detailed in Table r ≥ 0.4) for 12 of the 15 hypothesized relationships between pairs of domains or total scores (Table r = – 0.18) and Day 14 (r = – 0.35), the cTTP-PEQ Pain/Bruising domain and the MCMDM Bleeding Questionnaire Bruising Subscale score at baseline (r = 0.32), and the cTTP-PEQ Activity Limitation single-item domain and the WPAI-GH “impairment while working due to health” score at baseline (r = 0.38) and Day 14 (r = 0.30). Discriminant validity was confirmed for five of the seven hypothesized relationships with low correlations , the cTTP-PEQ Bruising/Pain domain and the NEI-FVQ-25 Near Vision and Distance Vision scales at baseline  and Day 14 , and the cTTP-PEQ Treatment Burden domain and the NEI-FVQ-25 General Vision and Distant Vision scales at Day 14 .Spearman’s rank correlation coefficients for the cTTP-PEQ domains and total score and the existing PRO measures are shown in Table es Table . However r = – 0.5, the cTVery high correlations (≥ 0.70) and very low correlations (≤ 0.19) that were not previously hypothesized were observed for the cTTP-PEQ domains and total score and other PRO measures at baseline and Day 14. These additional observed correlations are detailed in Table p ≤ 0.05) for all items except Items 4, 5, 6, 11, 12, 14, 15, 16, 19, 22, 24, and 26 at baseline and for all items, except Items 5, 6, 8, 17, 22, 24, and 26 at Day 14. The difference in cTTP-PEQ score between the two PGI-S groups was significant for all domains at baseline, with the exception of the single-item Activity Limitation score (p = 0.07) and single-item Headache score (p = 0.15), and for all domains at Day 14.Known-groups validity was evaluated by observing that the mean cTTP-PEQ scores generally increased with higher patient-reported impression of cTTP severity, as measured by categorized PGI-S score , for most items and domains at baseline and Day 14. The difference in mean cTTP-PEQ score between patients in the two PGI-S groups was significant (r = 0.79), the Mood score (r = 0.87), and the Treatment Burden score (r = 0.89). Individual items forming the multi-component domains showed moderate-to-high item-to-item correlation at baseline (Table S4) and Day 14 (Table S5).Item-to-total correlations for the cTTP-PEQ ranged from 0.32 (Item 12) to 0.75 (Item 18) at baseline and from – 0.03 (Item 26) to 0.81 (Item 8) on Day 14. The domain scores correlating the most with the total score were the Pain/Bruising domain  and 0.11 to 0.58 (Day 14) for the Pain/Bruising domain , from 0.39 to 0.54 (baseline) and 0.53 to 0.68 (Day 14) for the Cognitive Impairment domain , from 0.28 to 0.73 (baseline) and 0.50 to 0.79 (Day 14) for the Mood domain (Items 13 to 18), and from 0.22 to 0.86 (baseline) and 0.03 to 0.59 (Day 14) for the Treatment Burden domain . Item-to-item correlation was 0.72 (baseline) and 0.74 (Day 14) for the Visual Impairment domain (Items 9 and 10) (Table S4 and Table S5). Cronbach’s alpha was 0.88 at baseline and 0.91 on Day 14.Test–retest reliability is the assessment of the internal validity of a measure, it measures the consistency of results when the same test is repeated on the same sample at a different time. The measurement of a stable construct should remain stable over time. Test–retest reliability was assessed using ICCs  between two time points (Day 1 and Day 14) for subjects who reported no changes in health status between both assessments. The ICC ranged from 0.762 for the Mood domain to 0.943 for the Cognitive Impairment domain. The ICC for the cTTP-PEQ total score was 0.955  or mild (37.5%). Items with high floor effects (> 40%) included those measuring confusion (Item 7), blurry vision (Item 9), anxiety (Item 17), experience of mood swings (Item 18), activity limitation (Item 19), and worry due to problems relating to TTP treatment (Item 21). A direct consequence of a high floor effect is that such items under-discriminate and, as a result, are potentially not sensitive to change . It was Owing to the relatively small sample size, performing a factor analysis to assess the structural validity of the cTTP-PEQ was not possible. Originally, exploratory and confirmatory factor analysis was intended to be performed to investigate structural validity; however, this was not possible due to the nature and size of the sample distribution. The structure and domains of the proposed cTTP-PEQ instrument are based on a hypothetical conceptual model and framework, as well as item-to-item and item-to-total correlations. The cTTP-PEQ Pain/Bruising domain had the lowest support for its structural validity, with item-to-item correlations ranging from 0.05 to 0.63 at baseline and 0.11 to 0.58 on Day 14 and with moderate item-to-total correlations. This domain measures the worst pain experienced by the patient in the joints, muscle, abdomen, chest, and head in the past 24 h, and the worst bruising in the last 7 days. These low inter-item correlations may be explained by differences in the extent of thrombotic events at different locations in the body. Given certain domains have more items than others, and some items have a wider range of answer scores than others, the total score of the cTTP-PEQ could reflect a higher relative importance of domains with more items or items with higher possible scores. A factor analysis would have enabled a weighted sum of the measure, regardless of item numbers and score ranges per domain, but unfortunately the sample size did not permit these analyses.Convergent and discriminant validity of the cTTP-PEQ was confirmed by the observed correlations between domains. Moderate-to-high correlations were observed between the domains of the cTTP-PEQ and related concepts of the alternative PRO measures in the majority of hypothesized cases. However, there were three hypothesized relationships for which convergent validity was not supported: the cTTP-PEQ Visual Impairment domain and NEI-VFQ-25 Near Vision scale, the cTTP-PEQ Pain/Bruising domain and the MCMDM Bleeding Questionnaire Bruising Subscale score at baseline, and the cTTP-PEQ Activity Limitation single-item domain and the WPAI-GH “impairment while working due to health” score. The MCMDM Bleeding Questionnaire Bruising Subscale focuses on bruising, whereas the cTTP-PEQ Pain/Bruising domain focuses largely on pain. Similarly, the cTTP-PEQ Visual Impairment domain measures blurry vision and blind spots, whereas the Near Vision scale of the NEI-VFQ-25 focuses on loss of near vision. This conceptual reasoning could go some way to explaining the observed weaker correlation. A similar explanation can be found for the cTTP-PEQ Activity Limitation domain, which focuses on activity limitation in a general sense, correlating poorly with the WPAI-GH, which focuses on productivity loss and inability to participate in work-related activities.Test–retest reliability was assessed using ICCs  between two time points (Day 1 and Day 14) for subjects who reported no changes in health status between both assessments. While this is a common practice, it is potentially biased in that respondents self-select into the analysis set. Further, it ignores any measurement error associated with the anchor . Low correlations between the domain and total scores measuring unrelated concepts were confirmed for most hypotheses. The exceptions were the moderate correlations observed between the cTTP-PEQ Fatigue single-item domain and NEI-FVQ-25 Distant Vision scale, the cTTP-PEQ Bruising/Pain domain and NEI-FVQ-25 Near and Distant Vision scales, and the cTTP-PEQ Treatment Burden domain and NEI-FVQ-25 General Vision and Distant Vision scales. The extent to which the hypotheses were confirmed generally supports the convergent and discriminant validity of the cTTP-specific instrument.p ≤ 0.05) for all domains and the total score except the Activity Limitation and Headache domains at baseline. Single Items 4, 5, 6, 11, 12, 14, 15, 16, 19, 22, 24, and 26 at baseline and Items 5, 6, 8, 17, 22, 24, and 26 at Day 14, also did not support known-groups validity. These findings can be linked to the high floor effects described earlier and the generally low levels of patient-reported cTTP severity, as measured by the PGI-S. Therefore, known-groups validity was partially supported by the results. At the score level however, the mean cTTP-specific instrument scores for most domains, as well as the total score, increased with the increase of the patient-reported impression of cTTP severity, as measured by the PGI-S, and this difference was statistically significant, supporting known-groups validity (Table S6).Known-groups validity was assessed by comparing the mean scores of the cTTP-PEQ between patients separated on the basis of their PGI-S score. The differences in the cTTP-PEQ scores between the PGI-S normal and mild/moderate/severe groups were statistically significant  were receiving prophylactic treatment and did not modify their treatment during the course of the study. The control of cTTP symptoms by treatment may explain why most clinician- and patient-reported disease severity levels were mild. As such, patient responses were on the lower end of the scoring range, contributing to the aforementioned floor effects observed in this study. In addition, the small sample size prevented more robust assessment of the psychometric properties from being performed, and structural validity could not be assessed using a factor analysis. Therefore, item-to-item and item-to-total correlations were used to support the grouping of items, following the hypothesized conceptual model. In addition, a pragmatic scoring method was used to summarize individual items into domain scores. Therefore, the response options and number of items per domain will weight items differently using this approach. Considering the small sample size, it should also be noted that many of the bivariate correlation findings should be interpreted with caution, especially for the single items. The limited sample size also affects the feasibility of assessing the validity of single items as estimators. Future studies could aim to assess the cTTP-PEQ in patients with more severe disease to provide further insight on whether the observed floor effects are inherent to the sample or the PRO measure, and could allow the assessment of sensitivity to change. However, patients with severe disease are uncommon in the clinical environment due to the use of prophylactic treatment and would be challenging to recruit. Ideally, modern test theory methods would be used to further examine the alignment of the response options with patient severities and assess replicability of the findings. However, these methods would require larger sample sizes.Patients with the ADAMTS13 deficiency cTTP experience disease- and treatment-related complications and burdens that have a negative impact on their HRQoL , 10. AltBelow is the link to the electronic supplementary material.Supplementary Material 1


In this study, we investigated the contribution of LDTs to cell envelope synthesis in Pseudomonas aeruginosa grown in planktonic and biofilm conditions. We first assigned a function to each of the three P. aeruginosa LDTs by gene inactivation in P. aeruginosa, heterospecific gene expression in Escherichia coli, and, for one of them, direct determination of its enzymatic activity. We found that the three P. aeruginosa LDTs catalyze peptidoglycan cross-linking (LdtPae1), the anchoring of lipoprotein OprI to the peptidoglycan (LdtPae2), and the hydrolysis of the resulting peptidoglycan-OprI amide bond (LdtPae3). Construction of a phylogram revealed that LDTs performing each of these three functions in various species cannot be assigned to distinct evolutionary lineages, in contrast to what has been observed with PBPs. We showed that biofilm, but not planktonic bacteria, displayed an increase proportion of peptidoglycan cross-links formed by LdtPae1 and a greater extent of OprI anchoring to peptidoglycan, which is controlled by LdtPae2 and LdtPae3. Consistently, deletion of each of the ldt genes impaired biofilm formation and potentiated the bactericidal activity of EDTA. These results indicate that LDTs contribute to the stabilization of the bacterial cell envelope and to the adaptation of peptidoglycan metabolism to growth in biofilm.Peptidoglycan is an essential component of the bacterial cell envelope that sustains the turgor pressure of the cytoplasm, determines cell shape, and acts as a scaffold for the anchoring of envelope polymers such as lipoproteins. The final cross-linking step of peptidoglycan polymerization is performed by classical IMPORTANCE Active-site cysteine LDTs form a functionally heterologous family of enzymes that contribute to the biogenesis of the bacterial cell envelope through formation of peptidoglycan cross-links and through the dynamic anchoring of lipoproteins to peptidoglycan. Here, we report the role of three P. aeruginosa LDTs that had not been previously characterized. We show that these enzymes contribute to resistance to the bactericidal activity of EDTA and to the adaptation of cell envelope polymers to conditions that prevail in biofilms. These results indicate that LDTs should be considered putative targets in the development of drug-EDTA associations for the control of biofilm-related infections. N-acetylglucosamine (GlcNAc) and N-acetylmuramic acid (MurNAc) residues linked together by β-1,4 glycosidic bonds. In Gram-negative bacteria, the d-lactoyl group of MurNAc residues are linked to a conserved peptide stem containing an l-alanine at the first position (l-Ala1), a d-glutamic acid at the second position (d-Glu2), a diaminopimelic acid at the third position (DAP3), and two d-alanines at the fourth and fifth positions (d-Ala4 and d-Ala5). In Gram-positive bacteria, DAP3 is often amidated or replaced by an l-lysine (l-Lys3), which can be substituted by a short side chain .Peptidoglycan is a major component of the cell envelope that is present in almost all bacterial species. This complex heteropolymer is composed of linear glycan chains cross-linked by short stem peptides to form a three-dimensional network that surrounds the cytoplasmic membrane and provides resistance to the turgor pressure of the cytoplasm . The glyd-Ala4 in an acyl donor stem peptide to DAP3 in an acyl acceptor stem peptide. Synthesis of these so-called 4→3 cross-links is catalyzed by the d,d-transpeptidase activity of penicillin-binding proteins (PBPs) in two steps , which connect residues at the 3rd position of the donor and acceptor stems (3→3 cross-links)  . Similaridases 5–. LDTs anidases 5–. PBPs anidases 5––13.Enterococcus faecium selected in vitro , providing a link between the peptidoglycan and the outer membrane that is thought to contribute to the stability of the envelope , was found to exclusively display hydrolytic activity for cleavage of the amide bond connecting Lpp to tripeptide stems  and d-2-hyroxy  acids  acids . For thestrength .3–d-Ala4 amide bond. In Acinetobacter baumannii, a hydrolytic member of the LDT family acts as an l,d-carboxypeptidase, trimming off d-Ala4 of tetrapeptide stems and thereby contributing to peptidoglycan recycling  (l-Ala–d-iGlu–DAP–d-Ala [where iGlu is isoglutamic acid]), both in monomers and in the acceptor stem of dimers. The pentapeptide stem (l-Ala–d-iGlu–DAP–d-Ala–d-Ala) was not detected, indicating that the terminal d-alanines of stem pentapeptides that did not participate in peptidoglycan cross-linking were effectively trimmed off by d,d-carboxypeptidases. Cleavage of DAP–d-Ala amide bonds by l,d-carboxypeptidases or by endopeptidases generated the tripeptide stem l-Ala–d-iGlu–DAP. Most of the dimers (87%) contained 4→3 cross-links made by the d,d-transpeptidase activity of PBPs. The remaining dimers (13%) contained 3→3 cross-links formed by l,d-transpeptidases. Detection of the tripeptide l-Ala–d-iGlu–DAP linked to a Lys-Arg dipeptide (Tri→KR) revealed the anchoring of the OprI lipoprotein to the peptidoglycan . The disaccharide moiety was composed exclusively of GlcNAc and reduced MurNAc. Deacylated sugars, as previously reported  . Mass spreported , 40, wer with a conserved (S/T)XGCh(R/N) catalytic domain, in which C is the Cys nucleophile, X is any residue, and h is a hydrophobic residue . The function of these enzymes, designated LdtPae1 (PA14_54810), LdtPae2 (PA14_27180), and LdtPae3 (PA14_15840), was investigated by expressing the corresponding genes in a derivative of E. coli BW25113 obtained by deletion of the complete set of the six E. colildt genes   . These rPae2 in E. coli Δ6ldt led to the formation of an additional disaccharide-tripeptide monomer substituted by the dipeptide Lys-Arg (KR)  and P. aeruginosa (OprI) lipoproteins (24.8% identity) .Heterospecific expression of genes encoding LdtArg (KR) . This obPae3 in E. coli Δ6ldt did not lead to any modification of the muropeptide profile. Thus, LdtPae3 did not catalyze formation of 3→3 cross-links or the anchoring of the Braun lipoprotein in E. coli.Heterospecific production of LdtPae1ldt abolished the formation of 3→3 cross-linked dimers, indicating that LdtPae1 is the only peptidoglycan cross-linking l,d-transpeptidase produced by P. aeruginosa PA14 , confirming that this muropeptide originates exclusively from the anchoring of OprI to peptidoglycan. Together, these results   .Pae1, we produced a soluble fragment of LdtPae1 lacking the membrane anchor (residues 1 to 32) in E. coli and purified by metal affinity and size exclusion chromatography. Consistent with our previous results, LdtPae1 was functional in the formation of 3→3 cross-linked dimers  and carbapenem (meropenem) classes but not with the penam ampicillin. This β-lactam specificity was previously observed for LDTs from various bacteria  encoding an unregulated (p)ppGpp synthase (RelA′) and by the deletion of the chromosomally located relA gene encoding the wild-type (p)ppGpp synthase ppGpp -inducible promoter, in the derivative of E. coli BW25113 that overproduces the (p)ppGpp alarmone. Plasmids coding for inducible production of YcbB and YnhG were used as positive and negative controls, respectively. Production of P. aeruginosa LdtPae1 did not mediate β-lactam resistance in this host, as previously found for E. coli YnhG . These domains are likely to be essential for the bypass of PBPs by enabling YcbB to recruit accessory proteins required to carry out peptidoglycan cross-linking during the entire cell cycle. These accessory proteins may include the bifunctional transpeptidase-glycosyltransferase class A PBP1b, which displays affinity for YcbB ppGpp . We theroli YnhG . Thus, Yfor YcbB , and scafor YcbB , 42.P. aeruginosa PA14 and isogenic derivatives obtained by deletion of l,d-transpeptidase genes (Table S3). Deletion of ldtPae1, ldtPae2 plus ldtPae3, or of the three l,d-transpeptidase genes did not affect the susceptibility of P. aeruginosa to any of the tested antibiotics. Thus, the l,d-transpeptidases did not contribute to growth of PA14 in the presence of subinhibitory concentrations of β-lactams or to the efficacy of the permeability barrier mediated by the outer membrane.The activity of 31 antibiotics, including 20 β-lactams, was tested by the disk diffusion assay against E. coli, since this compound prevents the stabilization of the outer leaflet of the outer membrane by Mg2+ cations  is required for biofilm formation by an enteroaggregative strain of hia coli . This prPae1 and that this growth condition was associated with a highly cross-linked peptidoglycan. Growth in biofilm also favored the anchoring of OprI to peptidoglycan, indicating that LdtPae2 was active under this growth condition , indicating that the cell envelope of the mutants was fully compatible with the assembly of a functional flagellum.Our second objective was to determine whether the activity of the LDTs modulate biofilm formation. The deletion of te assay . The comPae1, a lipoprotein-anchoring activity was assigned to LdtPae2, and neither activity was assigned to LdtPae3 (see above) . More distantly related species, such as E. coli and P. aeruginosa or E. faecium and S. aureus (which does not harbor any LDT), produce various numbers of LDTs (0 to 6), and the domain composition is not conserved .A peptidoglycan cross-linking activity was assigned to Ldte above)  and 2. TE. coli LDTs (SXGChR versus SXGChA) and proposed that the latter motif was specific for enzymes hydrolyzing the amide bond connecting tripeptide stems to lipoproteins  . LdtPae2 (OprI anchoring) belonged to a lineage that included both E. coli Lpp anchoring  and peptidoglycan cross-linking (YnhG) enzymes. For LdtPae1 (formation of 3→3 cross-links), the highest levels of identity were observed for YciB from Bacillus subtilis , LdtMab3 from Mycobacterium abscessus , and YcfS of Campylobacter jejuni and E. coli . Together, these results imply that LDTs with lipoprotein anchoring and 3→3 cross-linking activities cannot be assigned to two divergent evolutionary lineages, because they are scattered in the various branches of the phylogram depicted in Fig. S3.We then evaluated whether LDTs catalyzing similar reactions could be identified based on the presence of specific sequence signatures. Winkle et al. identified a polymorphism in the conserved catalytic motif of E. coli) , 33. LdtPae1, which catalyzes the formation of 3→3 peptidoglycan cross-links, comprises an N-terminal bona fide LDT catalytic domain with a conserved catalytic motif (SHGCIR), followed by a related domain that lacks the catalytic Cys residue (QLGKIR) . These two domains are clustered in the same lineage, suggesting a duplication. This domain architecture has not been detected in any other LDT sequence. The role of the catalytically deficient domain LdtPae11 remains to be determined. It is tempting to speculate that it could be involved in peptidoglycan binding, a function mediated by unrelated domains in other LDTs , the anchoring of lipoprotein OprI to peptidoglycan (LdtPae2), and the hydrolysis of the resulting tripeptide→OprI amide bond (LdtPae3). The activity of these enzymes was investigated by in vitro assays for purified LdtPae1 (ldt genes from the chromosome of P. aeruginosa PA14 (ldt genes in E. coli (Pae1 and LdtPae2 in the heterologous host might be accounted for by the conserved structures of both the peptidoglycan subunit of P. aeruginosa and E. coli (Pae3 was tentatively assigned to the release of OprI based on both the absence of detectable amide bond-forming activity (formation of 3→3 and tripeptide→ OprI bonds) and the close similarity between LdtPae3 and E. coli hydrolase YafK.Active-site cysteine eactions . Here, w LdtPae1 , deletioosa PA14 , and het E. coli . The fun E. coli  and the ldt genes appear to belong to bacterial core genomes, they are not stably inherited in distantly related lineages. In contrast, comparison of high-molecular-weight PBPs involved in peptidoglycan cross-linking clearly identifies orthologues in distantly related lineages . LDTs with lipoprotein anchoring and 3→3 cross-linking activities cannot be assigned to two distinct evolutionary lineages. Thus, although lineages .ldt genes and of oprI has been explored using various assays. The deletion of these genes did not result in hypersusceptibility to antibiotics or impaired motility . These observations indicate that the permeability barrier of the outer membrane and the functionality of the flagellum were preserved in the mutants. Loss of OprI or of the anchoring of this lipoprotein to peptidoglycan led to a destabilization of the cell envelope, as revealed by the bactericidal effect of EDTA . Deletion of ldtPae1 had a modest but significant (P < 0.05) impact on the ability of P. aeruginosa PA14 to form biofilms  broth (LB) at 37°C. For quantitative analyses of the peptidoglycan of P. aeruginosa PA14 in the exponential phase of growth, bacteria were grown in 1 L of LB broth and collected at an optical density at 600 nm (OD600) of 0.8. For quantitative analyses of the peptidoglycan of P. aeruginosa PA14 grown in biofilm, continuous-flow biofilm microfermentors containing a removable glass spatula were used as described previously (600 of 1.0 (ca. 5.0 × 108 bacteria/mL) for 5 min. The spatula was placed into the microfermentor, and biofilm culture was performed at 37°C in Miller LB broth. The flow rate was adjusted so that the total time for renewal of microfermentor medium was shorter than the bacterial generation time, thus minimizing planktonic growth by constant dilution of nonbiofilm bacteria. Biofilms were allowed to grow on the glass spatula for 72 h, after which the microfermentor was vortexed for 1 min to resuspend the bacterial population. The resulting bacterial suspension (50 mL) was centrifuged for 15 min at 7,000 × g (4°C), and peptidoglycan was extracted by the hot SDS procedure. Peptidoglycan analyses were performed for a minimum of three biological repeats.For the analyses of the muropeptide composition of the peptidoglycan from eviously . Biofilm4, and purified by rpHPLC. The mass of muropeptides was determined on a Bruker Daltonics maXis high-resolution MS (Bremen Germany) operating in the positive mode, as previously described  was incubated with peptidoglycan fragments (50 μM) in 25 mM Tris-HCl (pH 8.0) at 20°C. LC-MS was performed with a Nucleoshell RP 18 column  coupled to a low-resolution LCQ Advantage mass spectrometer (ThermoElectron). The reactions were also carried out in the presence of additional d-Met (1 mM).Reduced disaccharide-tetrapeptide and lactoyl-tetrapeptide were extracted from the peptidoglycan of escribed . PurifiePae1 (10 μM) with β-lactams (100 μM) at 20°C in 5 mM Tris-HCl (pH 8.0) (The formation of drug-enzyme adducts was tested by incubating Ldt(pH 8.0) . Five mildt genes and oprI .The two-step allelic exchange procedure was used for deleting and oprI . BrieflyAntibiograms were performed in triplicate by the disk diffusion assay in cation-adjusted Mueller-Hinton agar plates according to the guidelines of the European Committee on Antimicrobial Susceptibility Testing (EUCAST) .600 of 0.9. EDTA was added , bacteria were incubated in 2-mL Eppendorf tubes at 37°C, and samples were withdrawn at 90 min, 180 min, and 270 min for viable cell counts on agar plates. Data were obtained in three biological repeats.Bacteria were grown overnight to the stationary phase in LB (Miller) broth at 37°C, washed twice in 50 mM Tris-HCl buffer (pH 8.0), and resuspended in the same buffer to a final ODAn isolated colony from an overnight agar plate was inoculated onto a soft agar plate containing tryptone broth (10 g/L), NaCl (5 g/L), and agar  . The plaFormation of biofilm was determined by crystal violet staining in 96-well microtiter plates, as previously described . A microt test was performed to analyze the relative abundance of dimers and 3→3 cross-links in the peptidoglycan of P. aeruginosa PA14. A Brown-Forsythe and Welch analysis of variance (ANOVA) test was performed to analyze the impact of ldt gene deletions on bacterial killing by EDTA and the quantification of biofilm formation. A P value of 0.05 was considered statistically significant in both types of tests.The data were analyzed using GraphPad Prism version 9 . An unpaired E. coli , P. aeruginosa , Salmonella enterica serovar Typhimurium , Campylobacter jejuni (Cje_YcfS and Cje_Ybis), Neisseria meningitidis (LdtNme), Bacillus subtilis , Clostridioides difficile , Mycobacterium tuberculosis (LdtMt1 to LdtMt6), Mycobacterium abscessus (LdtMab1 to LdtMab6), Enterococcus faecium (Ldtfm), Enterococcus faecalis (Ldtfs), and Coxiella burnettii (LdtCbu2). The sequences of the domains were aligned using Clustal Omega , providing the identity matrix reported in Table S5. The phylogram reported in Fig. S3 was obtained on iTOL (Forty YkuD-related domains were retrieved from the genome of 12 bacterial species, including  on iTOL .


Background: Acute decompensated heart failure (ADHF) is a highly prevalent diagnosis in the emergency department and is associated with high morbidity and mortality. As mortality remains high even in patients discharged from the emergency, it becomes mandatory to identify markers predictive of mortality in order to guide the disposition of such patients. No literature is available on the prognostic significance of Troponin I in ADHF patients in an emergency setting from an Indian standpoint.Objectives: This study was aimed at identifying the ability of Troponin I levels at presentation to predict one-month mortality in patients with ADHF.Methods: The study was conducted in the emergency department of a tertiary healthcare center in north India. Serum cardiac Troponin I (cTnI) levels at presentation were assayed in 101 patients and a one-month follow-up was done.Results: cTnI levels were > 0.02 ng/mL in 51 patients (50.5%). ROC analysis showed an accuracy of 63% in predicting mortality (p < 0.05). Univariate and multivariate analysis showed an OR of 2.58 and 2.74, respectively , suggesting cTnI to be a significant predictor of mortality in ADHF. N-terminal proBNP (NT-proBNP)  and left ventricular ejection fraction  were not found to be significant predictors of mortality on regression analysis.Conclusion: cTnI levels at presentation are a significant predictor of short-term mortality in ADHF and can be used in an emergency setting to guide treatment, disposition, and follow-up plans of these patients. Heart failure (HF) is defined as symptoms or signs due to abnormalities of cardiac structure or function, with the diagnosis supported by increased natriuretic peptide levels as well as evidence of systemic or pulmonary congestion . Acute dDifferent markers have been postulated to show an association with poor prognosis in patients with HF. Missov et al. provided the first evidence of cardiac troponin-I (cTn-I) as a potentially sensitive and specific marker of myocardial injury in HF . The higThis was a single-center, prospective, observational study done in the ED of a tertiary care teaching hospital in north India. The study was conducted from August 2020 to June 2022, after obtaining ethical approval from the Institute Ethics Committee (IECPG-290/22.07.2020).Adult patients (age > 18 years) presenting to the ED with ADHF, were recruited in the study. ADHF was defined as any sudden onset or sudden worsening of symptoms of heart failure (HF) within seven days, HF being defined by the Framingham criteria . PatientData were collected in a predesigned questionnaire. Data collected included demographic profile, presenting symptoms of the patient, previous medical/surgical history, and physical examination findings. Five milliliter blood sample was drawn from the patient at presentation and collected in standard dipotassium edetate vials for Troponin I and NT-proBNP assay. Estimation was done using Radiometer AQT90 Flex Analyzer . Reference ranges for Troponin I and NT-proBNP were <0.02 ng/mL and 70-133 pg/mL, respectively. Bedside echocardiography using Fujifilm Sonosite Ultrasound machine , was done to determine Left ventricular ejection fraction (EF). The investigator was well trained in the EF assessment in ED prior to the start of the recruitment of study participants. Other investigations included electrocardiograms, chest radiography, serum electrolytes, and serum creatinine. The endpoint of the study was one-month mortality, and this information was collected by contacting the patient or his/her family members by phone one month after the presentation.Quantitative variables were presented as mean with standard deviation (SD)  and median with interquartile range (IQR) . Qualitative variables were expressed as counts and percentages. The receiver operating characteristics (ROC) curve was used to assess the accuracy of cTn-I, NT-proBNP, and EF values in predicting mortality, and to determine valid cut-offs by the Youden’s-J test. Logistic regression (LR) was used to analyze the predictors of one-month mortality where covariates included demographic parameters, vitals at clinical presentation, cTn-I, NT-proBNP, and EF. The cTn-I, NT-proBNP, and EF values were converted to categorical values using the cut-offs from ROC analysis and entered into the LR model. Only the covariates that were significant predictors of one-month mortality in the univariate analysis were considered for the multivariate LR model. Statistical significance was set at a two-tailed p-value of less than 0.05. The data were collected in Microsoft Excel  and all statistical analyses were done with IBM SPSS Statistics for Windows .A total of 208 patients presenting with ADHF were screened for the study. Eighty-eight patients were excluded: 24 patients had ACS or myocarditis at presentation, 54 had underlying valvular heart disease and 10 had CHD. Nineteen out of the 120 patients could not be followed up mostly due to logistical reasons owing to the COVID-19 pandemic, resulting in a final sample size of 101 patients for analysis  being male. The most common presenting symptom was shortness of breath (97%) followed by swelling of feet (37.6%) and cough with expectoration (30.7%). Most common comorbid conditions in the studied population were hypertension (50.5%) and diabetes mellitus (35.6%). The detailed demographic characteristics, vitals at presentation and laboratory parameters of the study population are presented in Table The mean values of cTn-I, NT-proBNP and EF were 0.195 ± 0.50 ng/mL, 16822.1 ± 12954.6 pg/mL and 35 ± 13%, respectively. Mean cTnI values were significantly higher in patients who died as compared to those who were alive  at one-month follow-up. Similarly, the mean NT-proBNP was significantly higher in the former group as compared to the latter . The mean EF tended to be lesser in patients who died at one month, but the difference was not statistically significant . Detailed comparison is depicted in Table The ROC curve was prepared to assess the ability of the individual markers to predict mortality and shown in Figure The AUROC of cTn-I for predicting one-month mortality was 0.625 . A cTn-I value of more than 0.020 ng/mL (cut-off decided by Youden’s-J test) was found to be 66.7% sensitive and 56.3% specific for prediction of mortality. NT-proBNP showed an AUROC of 0.654 . An NT-proBNP cut-off of 10,000 pg/mL showed a sensitivity of 70% and a specificity of 52.11% for prediction of mortality. ROC analysis of EF showed an AUROC of 0.627 . A cut-off of 40% was shown to have a sensitivity of 76.66% and a specificity of 38.28% for predicting one-month mortality. None of the markers were significantly superior to the others in predicting mortality as per ROC analysis (p > 0.05). The diagnostic statistics of all parameters in predicting study outcome are reported in Table On logistic regression, the covariates that showed a significant association for with mortality were cTn-I and NT-proBNP. A cTn-I value of more than 0.020 ng/mL was found to have an odds ratio (OR) of 2.58  in predicting mortality. An NT-proBNP value of more than 10,000 pg/mL predicted mortality with an OR of 2.54 . However, the EF was not able to predict the mortality significantly . Subsequent multivariate analysis showed only cTn-I to be a significant predictor of mortality . The results of univariate and multivariate logistic regression analysis are shown in Table The mechanisms describing the elevation of cardiac CTn-I in patients of HF are still open to debate. The commonly postulated mechanisms that occur in HF include sub-endocardial ischemia resulting in necrosis of the myocytes ,12, incrWe conducted this prospective observational study to investigate the association of different markers like cTn-I with one-month mortality in ADHF patients in Indian settings. Our study population showed similarities in demographic characteristics to another Indian HF registry . In our The cTn-I was elevated (> 0.02 ng/mL) in 50.5% of patients recruited in our study. The ROC analysis showed that cTn-I was 63% accurate in predicting mortality. Arenja et al. found that the cTn-I was raised in 41% of 357 AHF patients (cutoff of 0.028 ng/mL) and cTn-I was 74% accurate in predicting mortality. This discrepancy, however, could be explained by the fact that, unlike our study, this study included patients with ACS. The OR and adjusted OR of raised cTn-I (> 0.02 ng/mL) in predicting mortality were 2.58 and 2.74 in our study, respectively, which were in line with a similar study by Peacock et al. based on the ADHERE database . They coIn our study, ROC analysis of NT-proBNP showed an accuracy of 65% in predicting mortality (p - 0.016). Taking a cut-off of 10,000 pg/mL gave a sensitivity of 70% and a specificity of 52% in predicting mortality. While raised NT-proBNP appeared to predict mortality significantly on univariate analysis, multivariate logistic regression did not provide a significant correlation with one-month mortality. Noveanu et al.  found NTIn our study, the mean EF in the group of patients who survived at one month was not significantly different from those who did not. An ejection fraction of less than 40% was not found to significantly predict mortality on logistic regression. Bhatia et al. studied 2,802 patients of AHF patients and found no significant difference in mortality rates between the HFpEF  and HFrEF (HF with reduced EF of less than 40%) groups at 30 days . SimilarThere are many novel biomarkers that are being postulated to be predictive of mortality in ADHF. Some of them include soluble suppression of tumorigenicity-2(sST2), galectin-3, copeptin, MR-proAdrenomedullin, and high sensitivity C-reactive protein. Studies show sT2 to be a significant, independent predictor of both cardiovascular and all-cause mortality in ADHF -29.Our study had its share of limitations. A sample size of 100, while establishing a clear correlation between cTn-I and mortality in ADHF, was probably not enough to identify other probable predictors of mortality. Serial assessments of Troponin I and NT-proBNP were not done and their prognostic significance was not assessed in this study. Other suggested risk factors for all-cause in mortality include NYHA class IV presentation, lower systolic blood pressure, serum creatinine, and oxygen saturation at presentation . As therIn an emergency setting, Troponin I is a significant predictor of one-month mortality and serves better than NT-proBNP or left ventricular ejection fraction for the same, in patients with ADHF. cTnI values at presentation can hence be used to guide treatment, disposition, and follow-up decisions for these patients from the ED.


Purpose: To assess functional and anatomical outcomes of intravitreal anti-Vascular Endothelial Growth Factor (anti-VEGF) monotherapy versus combined with verteporfin Photodynamic Therapy (PDT) for Retinal Angiomatous Proliferation (RAP).Methods: Studies reporting outcomes of intravitreal anti-VEGF monotherapy and/or in combination with verteporfin PDT in RAP eyes with a follow-up ≥ 12 months were searched. The primary outcome was the mean change in best corrected visual acuity (BCVA) at 12 months. Mean change in central macular thickness (CMT) and mean number of injections were considered as secondary outcomes. The mean difference (MD) between pre- and post-treatment values was calculated along with 95% Confidence Interval (95% CI). Meta-regressions were performed to assess the influence of anti-VEGF number of injections on BCVA and CMT outcomes.Results: Thirty-four studies were included. A mean gain of 5.16 letters (95% CI = 3.30–7.01) and 10.38 letters (95% CI = 8.02–12.75) was shown in the anti-VEGF group and combined group, respectively . A mean CMT reduction of 132.45 µm (95% CI = from −154.99 to −109.90) and 213.93 µm (95% CI = from −280.04 to −147.83) was shown in the anti-VEGF group and combined group, respectively . A mean of 4.9 injections (95% CI = 4.2–5.6) and 2.8 injections (95% CI = 1.3–4.4) were administered over a 12-month period in the anti-VEGF group and combined group, respectively. Meta-regression analyses showed no influence of injection number on visual and CMT outcomes. High heterogeneity was found across studies for both functional and anatomical outcomes.Conclusion: A combined approach with anti-VEGF and PDT could provide better functional and anatomical outcomes in RAP eyes compared with anti-VEGF monotherapy. Retinal angiomatous proliferation (RAP) was firstly described by Yannuzzi et al. as a distinct form of neovascularage-related macular degeneration (nAMD). .According to the anatomic classification, RAP is defined as “type 3 neovascularization”. . The pecIntravitreal anti-vascular endothelial growth factor (anti-VEGF) therapy has become the first line treatment for nAMDand for RAP lesions as well. . On the On this basis, intravitreal anti-VEGF therapy has been used in combination with photodynamic therapy (PDT) in attempt to achieve a better control of RAP lesions. . This coThe purpose of the present systematic review with meta-analysis was to collect available evidence on intravitreal anti-VEGF therapy alone or combined with PDT in RAP eyes and to assess whether combining anti-VEGF therapy with PDT could have a synergic effect and lead to better functional and anatomical outcomes.The study was conducted according to the guidelines of the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) group  to include eyes affected by retinal angiomatous proliferation that were treated with intravitreal anti-VEGF therapy alone  and/or in combination with photodynamic therapy; 2) to have a follow-up of at least 12 months; 3) to report visual and/or anatomical outcomes. Case reports and case series with less than 10 cases were excluded. Choroidal neovascular membranes different from RAP were excluded. When clarifications for study eligibility were needed, we contacted study’s authors.Eyes treated with anti-VEGF therapy alone were included in the anti-VEGF group, while eyes treated with anti-VEGF therapy combined with PDT were included in the combined group.The primary outcome of interest was the mean change in best corrected visual acuity (BCVA) in the two groups. Mean change in central macular thickness (CMT) on optical coherence tomography (OCT) was considered as secondary outcome. The influence of the number of injections on BCVA and CMT in either group was considered a secondary outcome as well. Central macular thickness referred to the average thickness of the fovea-centered area with 1 mm diameter.Two investigators (MF and IM) evaluated independently the eligibility of identified studies. The same two investigators (MF and IM) analyzed and extracted data from each included study in an independent fashion. A third investigator (VB) was involved in case of disagreement. The following items were collected from each included study: first author, publication year, country, study design, number of eyes, mean age, type of treatment, follow-up. For both the anti-VEGF group and the combined group, the following data were collected: number of eyes, mean age, naïve/non-naïve status, type of anti-VEGF drug and treatment protocol, number of injections, BCVA change, CMT change, follow-up. Information on type of PDT protocol, namely, standard verteporfin PDT, , half-doRisk of bias of randomized trials was evaluated by the means of the Cochrane collaboration tool. . Risk of2 index and the Q-statistics were used to measure and test heterogeneity across studies. When a significant heterogeneity was found (I2 > 50% and Q-statistics p < 0.1), a random effect model was fitted applying the DerSimonian-Laird method. Subgroup analyses were conducted to compare BCVA and CMT outcomes between the anti-VEGF group and the combined groups. Meta-regressions were performed to assess the influence of anti-VEGF number of injections on BCVA and CMT outcomes. Results of the meta-regressions were reported as β coefficient and its standard error (SE). Publication bias was tested using the Egger’s test and by visual inspection of funnel plots’ symmetry. Analyses were conducted on STATA (version 17) and were two-tailed, with a level of statistical significance α < 0.05.For BCVA and CMT change, pooled effect size was investigated through meta-analysis and mean difference (MD) between pre- and post-treatment values was reported along with 95% Confidence Interval (95% CI). The IThe flow diagram of the study selection is illustrated in versus combined with PDT. . In the anti-VEGF group, a mean gain of 5.16 letters was shown at 12 months (95% CI = 3.30–7.01). A significant heterogeneity was found across studies included in the anti-VEGF group . In the combined group, a mean gain of 10.38 letters was found at 12 months (95% CI = 8.02–12.75). Heterogeneity was low in the combined group . Of note, visual gain in the combined group was higher compared with the anti-VEGF group  and 95% CI of the combined group does not overlap with those of the anti-VEGF group. Accordingly, the test of group differences revealed that 12-month visual gain in the combined group was significantly greater compared with the anti-VEGF group (p < 0.01).Data from 22 and 9 studies were pooled together to explore mean visual change at 12 months in the anti-VEGF group and in the combined group, respectively . Overall2 = 93.55%; p < 0.01). In the anti-VEGF group, a mean CMT reduction of 132.45 µm was found at 12 months (95% CI = from −154.99 to −109.90). A significant heterogeneity was shown across studies included in the anti-VEGF group . In the combined group, a mean CMT reduction of 213.93 µm was evident at 12 months (95% CI = from −280.04 to −147.83). Heterogeneity was high in the combined group as well . Of note, at 12 months a greater CMT reduction was shown in the combined group compared with the anti-VEGF group  and 95% CI of the combined group does not overlap with those of the anti-VEGF group. Accordingly, the test of group differences demonstrated a significantly greater CMT decrease in the combined group compared with the anti-VEGF group (p = 0.02).Data from 16 and 7 studies were pooled together to explore mean CMT change at 12 months in the anti-VEGF group and in the combined group, respectively . Overallp = 0.02).The average number of injections over a 12-month follow-up was higher in the anti-VEGF group compared with the combined group: a mean of 4.9 injections (95% CI = 4.2–5.6) were administered in the anti-VEGF group while a mean of 2.8 injections (95% CI = 1.3–4.4) were administered in the combined group  lies in a synergic mechanism of action of these two therapeutic approaches. In fact, PDT could induce complete occlusion of the retinal–retinal anastomosis, while intravitreal anti-VEGF therapy could counteract the release of VEGF caused by the PDT-related hypoxia in the choriocapillaris. .versus intravitreal anti-VEGF therapy combined with PDT in RAP eyes.  was yet superior to the maximum improvement in the anti-VEGF alone group (6.56 letters), confirming that the combined approach yielded better visual results.With regard to macular thickness, the results of the two randomized trials showed a comparable final CMT between eyes treated with anti-VEGF monotherapy and eyes treated with anti-VEGF therapy combined with PDT. . ConversOur findings showed that combined therapy with PDT and intravitreal anti-VEGFs could yield better outcomes with a lower number of injections, thanks to their different and synergic mechanism of action.This may also have a positive impact on the financial burden, on the anxiety of patients and reduce the risk of injection-related complications and side effects. .In the present meta-analysis, combined therapy has also shown a good safety profile despite PDT being used in all studies except for one with standard dose and fluence.In light of a widespread use of PDT at lower dose and fluence, further studies are needed to investigate efficacy and safety of a combined approach using modified PDT protocols  for RAP lesions.In the present meta-analysis, no study on brolucizumab was included. There is paucity of data in literature on the use of brolucizumab in the treatment of RAP lesions. Only a retrospective case series on 12 eyes showed a good short-term efficacy of brolucizumab in reducing the size of type 3 neovascular membranes. .The present study presents some limitations. First, significant heterogeneity was found among included studies. This could limit the strength of our findings. A possible reason for high heterogeneity could be the variability in clinical characteristics and treatment protocols between included studies. However, all studies based RAP diagnosis on fluorescein and indocyanine green angiography and most included studies adopted a protocol treatment based on 3 monthly injections followed by a pro re nata regimen. Second, only two randomized clinical trials were included in this systematic review, of which only one provided data included in our pooled analyses. Furthermore, quantitative analyses were carried out from tabulated data extracted from each study because no individual data was available. However, confidence intervals yielded by meta-analysis studies are more powered and more accurate compared with individual studies. . Finally


Tajikistan has a high burden of rifampicin-resistant TB (RR-TB), with 2,700 new cases estimated for 2021 . TB is spread among household members through close interaction and children exposed through household contact progress to disease rapidly and frequently.We retrospectively analysed programmatic data from household contact tracing in Dushanbe over 50 months. We calculated person-years of follow-up, contact tracing yield, number needed to screen (NNS) and number needed to test (NNT) to find one new case, and time to diagnosis.We screened 6,654 household contacts of 830 RR-TB index cases; 47 new RR-TB cases were detected, 43 in Year 1 and 4 in Years 2 or 3. Ten were aged <5 years; 46/47 had TB symptoms, 34/45 had chest radiographs consistent with TB, 11/35 were Xpert Ultra-positive, 29/32 were tuberculin skin test-positive and 28/47 had positive TB culture and phenotypic drug susceptibility results. The NNS to find one RR-TB case was 141.57 and the NNT was 34.49. The yields for different types of contacts were as follows: 0.7% for screened contacts, 2.9% for tested contacts, 17.0% for symptomatic contacts and 12.1% for symptomatic contacts aged below 5 years.RR-TB household contact tracing was feasible and productive in Tajikistan, a low middle-income country with an inefficient healthcare delivery system. TB contact tracing, especially among household contacts of TB index cases, has been shown to be an effective strategy for identifying those at highest risk of TB infection and active disease. This ensures that 1) diagnostics and treatment can be initiated early in active disease, 2) latent TB infection can be identified and treated with TB preventive therapy (TPT) in drug-susceptible TB (DS-TB) contacts, 3) vulnerable DS-TB contacts without evidence of infection  can be given TPT to prevent active disease, and 4) TPT can be considered for vulnerable drug-resistant TB (DR-TB) contacts and those with TB infection.5Early detection and proper treatment of TB disease reduces transmission, morbidity and mortality. However, the most recent WHO World TB Report estimates that there are substantial proportions of undiagnosed, untreated pulmonary TB cases in communities.7Although contact tracing of high-risk groups is widely included in global and national policies, including Tajikistan’s national TB guidelines, this activity is rarely prioritised outside of low-burden, resource-rich settings despite its benefits for reducing TB community transmission.13The average household size in Tajikistan in 2021 was 5.56 persons;15Tajikistan guidelines dictate symptoms screening for all DS-TB and DR-TB household contacts, followed by diagnostic testing of contacts less than 5 years of age, contacts with HIV, and contacts aged ≥5 years with TB symptoms.The Ministry of Health (MOH) has recently called for intensified efforts to scale up contact tracing and ACF through a systematic approach at the primary healthcare level, in polyclinics , and involving general practitioners, paediatricians and community healthcare workers in rural areas.9A 2016 review of DS-TB contact tracing in Dushanbe found successful treatment outcomes and TPT coverage. However, they observed that there is potential to enhance the scale of ACF and the implementation of national protocols by developing effective strategies, including the establishing of data linkages to enable routine monitoring and evaluation of the contact tracing process to ensure its quality and effectiveness.16In this study, we sought to evaluate household contact tracing in Dushanbe, focusing on the yield, rate of progression following exposure and risk factors associated with a TB diagnosis.We conducted a retrospective cohort study to analyse routinely collected programme data from Dushanbe, Tajikistan, and surrounding districts  for 50 months (November 2017–December 2021). The study cohort included all household contacts of index patients, regardless of age, diagnosed with RR-TB during the study period. DS-TB contacts were excluded from the study. Household contact tracing data was linked to treatment registers and laboratory databases using a unique identifier.The primary objective of our study was to describe the yield and efficiency of household contact tracing of RR-TB index cases performed according to Tajikistan national guidelines. In those referred for diagnostic testing, we wanted to know how many were subsequently diagnosed and treated for RR-TB in various subgroups. Our secondary objectives included the following parameters: 1) time to diagnosis, 2) person-years of follow-up, 3) the number needed to screen (NNS) to find a new RR-TB case, and 4) the number needed to test (NNT) to find a new RR-TB case.Our study started in 2017 and all positive drug susceptibility testing (DST) results were recorded from patients enrolled prior to 2021; we therefore used pre-2021 WHO definitions of MDR-TB, RR-TB and extensively drug-resistant (XDR-TB).17We followed the Tajikistan national guideline for contact tracing and TB testing .9 Contac9Our primary analysis describes the yield of household contact tracing overall , by age, sex, presence or absence of TB symptoms, and in those with follow-up testing. In the secondary analysis, we determined the following: 1) person-years of follow-up: total days from earliest to last evaluation for all contacts who agreed to participate divided by 365; 2) the time from first assessment to disease progression: days from the first evaluation of those who agreed to participate to RR-TB confirmation; 3) the NNS: number of contacts screened divided by the number of new RR-TB cases identified; and 4) the NNT: number of contacts with follow-up testing divided by the number of new RR-TB cases identified.All household contacts of RR-TB patients identified in participating MOH clinics in Dushanbe and surrounding districts who agreed to 6-monthly follow-up for 3 years were eligible.Households that declined a visit and contacts whose record did not contain information on the index case or DST results were excluded.This study met the exemption criteria for ethical review as a retrospective review of routinely collected data of an activity as per the Tajikistan national TB guidelines.We visited 6,654 household contacts of 830 RR-TB index cases (average: 8.0 contacts/case) at least once from 2017 to 2021. The baseline characteristics of all contacts are shown in We identified 47 new active RR-TB cases from 38 index cases, totalling over 2,908 person-years of follow-up . The aveAmong 47 new RR-TB cases identified out of 6,654 contacts, the mean time from first assessment to disease progression and confirmation was 67 days (standard deviation: ±135). The average age of a new case was 11.8 years and over three-quarters were under 14 years of age. All but one patient had TB signs/symptoms. That patient was a 9 year-old female who was tested because of clinical suspicion and had a positive TB culture for XDR-TB. None of the 1,280 patients aged <5 years without TB signs/symptoms were confirmed with TB disease. Twenty-eight of the 47 new RR-TB cases had DST results: 16 were MDR-TB and 12 had XDR-TB. Seven of the 12 XDR-TB cases would be diagnosed as pre-XDR-TB according to the new classification system. Twenty-three patients with a positive TB culture were Ultra-negative. Of the 19 cases without a positive TB culture, four had a positive Ultra assay. The remaining 15 incident cases with a negative Ultra assay and negative TB culture were diagnosed on clinical and epidemiologic grounds by the Tajikistan TB Consilium of experts. Only two participants were known to be HIV-positive but neither were diagnosed with RR-TB. The HIV prevalence in Tajikistan is estimated to be 0.2%.The screening process yielded an overall rate of 0.7%. Among the contacts subjected to further testing, the yield was 2.9%, while those with TB symptoms had a yield of 17.0%. Specifically, contacts aged below 5 years with symptoms showed a yield of 12.1%, while those aged ≥5 years with symptoms had a yield of 19.4%. The NNS was 141.57 and the NNT was 34.49. Twenty-four cases were diagnosed at first assessment and 43 cases within the first year. All new RR-TB patients were treated .Key study yields were as follows: 12.1% in symptomatic <5-year-old household contacts of index patients with RR-TB and 0.0% in <5 years household contacts without signs/symptoms. Our study findings are consistent with the peer-reviewed literature, but ours is the first such report from Central Asia and we had more contacts per case (8.0) than most other studies.26We reviewed four systematic review and meta-analysis publications.27New England Journal of Medicine found an overall yield of 1.79% for ACF compared with 0.75% for passive case-finding.27Our overall yield was 0.7%, which is lower than those in the studies we reviewed, which had an average overall yield of 2.50 (range 0.96–6.50).We found a yield of 12.1% for children aged <5 years with signs/symptoms. A study from Myanmar among household contacts of index patients with RR-TB reported a yield of 10.0% for children aged <5 years.We found a yield of 2.9% in contacts with further diagnostic testing, including 17.0% for all contacts with signs/symptoms and 19.4% for ≥5 years contacts with signs/symptoms. Comparisons to the literature are difficult, as most studies that we reviewed included some tests in their initial screening strategy and additional tests at the next step. However, Honjepari et al. reported a yield of 5.8% in all contacts with symptoms before further testing.24The number of household members per index patient with RR-TB in our cohort was higher (8.0) than that reported in the last Tajikistan government survey in 2021 (average: 5.6) and higher than seven publications that we reviewed .There are several limitations to our study. We did not have the full results of diagnostic testing in all new RR-TB cases, and no information on total or negative HIV tests and the site of TB disease (pulmonary vs. extrapulmonary). We did not record information on contacts who declined to participate; however, our contact tracing teams agreed this number was low. Our teams estimated that two-thirds to three-quarters of families agreed to home visits for contact tracing and that visits were appreciated. The acceptability of this form of TB investigation has been previously documented at 91.1%.Finally, we would like to highlight that the Ultra sputum assay missed several cases that had a positive TB culture and the TB culture missed a few Ultra-positive cases. Both tests should be performed in this vulnerable group.ACF through household contact tracing is important in settings such as Tajikistan as it serves to identify persons with RR-TB at an early stage, especially children less than 5 years of age, so that prompt and appropriate treatment can be initiated and advancing disease and complications can be limited. Our study also serves to validate WHO guidelines with respect to the key signs and symptoms in TB contacts and the most productive follow-up tests. RR-TB household contact tracing was an effective, feasible and productive intervention following national guidelines in Tajikistan, a low-income country with an inefficient healthcare delivery system. We believe that this intervention can and should be prioritised in similar settings.


Atrial fibrillation (AF) leads to intracardiac thrombus and an associated risk of stroke. Phase-contrast cardiovascular magnetic resonance (CMR) with flow-encoding in all three spatial directions (4D-flow) provides a time-resolved 3D volume image with 3D blood velocity, which brings individual hemodynamic information affecting thrombus formation. As the resolution and contrast of 4D-flow are limited, we proposed a semi-automated 4D-flow segmentation method for the left atrium (LA) using a standard-of-care contrast-enhanced magnetic resonance angiography (CE-MRA) and registration technique.LA of 54 patients with AF were segmented from 4D-flow taken in sinus rhythm using two segmentation methods. (1) Phase-contrast magnetic resonance angiography (PC-MRA) was calculated from 4D-flow, and LA was segmented slice-by-slice manually. (2) LA and other structures were segmented from CE-MRA and transformed into 4D-flow coordinates by registration with the mutual information method. Overlap of volume was tested by the Dice similarity coefficient (DSC) and the average symmetric surface distance (ASSD). Mean velocity and stasis were calculated to compare the functional property of LA from two segmentation methods.LA volumes from segmentation on CE-MRA were strongly correlated with PC-MRA volume, although mean CE-MRA volumes were about 10% larger. The proposed registration scheme resulted in visually successful registration in 76% of cases after two rounds of registration. The mean of DSC of the registered cases was 0.770 ± 0.045, and the mean of ASSD was 2.704 mm ± 0.668 mm. Mean velocity had no significant difference between the two segmentation methods, and mean stasis had a 3.3% difference.The proposed CE-MRA segmentation and registration method can generate segmentation for 4D-flow images. This method will facilitate 4D-flow analysis for AF patients by making segmentation easier and overcoming the limit of resolution. With an injection of gadolinium contrast agent, which shortens the T1 relaxation time, the image of the vascular system is acquired in 3D with enhanced resolution (−1 mm). Moreover, CE-MRA can benefit from the optimal timing of contrast agent injection. Therefore, fine structure in the heart can be readily recognized with CE-MRA without the need for additional CMR sequences.This study proposes a semi-automated segmentation method for LA by using enhanced contrast and resolution of the CE-MRA image. We hypothesized that the registration of CE-MRA and PC-MRA would transform segmentations from CE-MRA to 4D-flow coordinates, and this transformed segmentation can yield compatible analysis results from the conventional PC-MRA segmentation method.2.2.1.In this retrospective study, a total of 108 patients with paroxysmal AF with normal systolic function, scanned between June 2017 and January 2020 with a 4D-flow scan, were retrospectively included from the Cardiovascular Imaging Registry of Calgary (CIROC) database . There wA commercial software  was used to coordinate routine capture of patient informed consent and health questionnaires and for standardized collection of MRI-related variables. Patients with significant mitral or aortic valve disease and inappropriate/incomplete image reconstruction cases were not included. The study was approved by the University of Calgary's Conjoint Health Research Ethics Board, and all subjects gave written informed consent. All performed research activities were in accordance with the Declaration of Helsinki.2.2.All subjects were required to be in sinus rhythm at the time of CMR imaging. Patients and healthy subjects underwent an identical standardized MRI protocol using 3 T MR scanners Skyra/Prisma  inclusive of standard multi-planar steady-state free-precession (SSFP) cine imaging in 4-chamber, 3-chamber, 2-chamber, short-axis of the LV at end-expiration. A three-dimensional magnetic resonance angiography (MRA) of LA was performed using the administration of 0.2 mmol/kg gadolinium contrast  at 2–3 ml/s. Three volumetric acquisitions were performed: one pre-contrast (for subtraction), one during the first pass, one after contrast administration. Subtraction volume was used for LA segmentation. Acquisition parameters were: Acquisition matrix = 384 × 286; repetition time = 3.39 ms; echo time = 1.16; flip angle = 20–25 degrees; and spatial resolution = 1.17 × 1.17 × 1.19–1.39 mm.Approximately 5–10 min following contrast injection, 4D-flow MRI using an ECG retrospectively triggered sequence with respiratory navigator-based gating (WIP 785A) was taken with imaging parameters previously described , 18, 22:2.3.A commercial software  was used to determine the heart function from standard ECG-gated cine images. The short axis cine images were used to obtain LV end-diastolic volume (EDV), LV end-systolic volume (ESV), LV stroke volume (SV), LV mass, LV cardiac output (CO), LVEDV indexed to BSA, LVESV indexed to BSA, LV mass indexed to BSA and LV ejection fraction (LVEF).4D-flow MRI pre-processing was performed using an in-house program developed in Matlab. Preprocessing steps included noise masking, velocity anti-aliasing, and corrections for Maxwell terms and eddy currents –24.2.4.i is the measured time frame within the cardiac cycle out of a total number of N frames. Magntide and velocity images are noted by v, velocity encoding direction is given by x, y, z. LA was segmented slice-by-slice manually using in-house software written in Matlab .All data underwent preprocessing to correct for Maxwell terms, eddy current-induced phase offset, and velocity aliasing, if necessary. PC-MRA was generated from 4D-flow MRI by multiplying the magnitude image and the magnitude of velocity from the phase image for each voxel .PCMRA module  was usedFinally, inspection-passed segmentations were transformed and resampled to be conformed with 4D-flow coordinates using in-house software written in Matlab. Nearest-neighbor interpolation method is used in resampling to generate binary mask.2.6.The volume of the LA was measured from segmentations from PC-MRA and CE-MRA, respectively. The LA volume from CE-MRA is measured after transformation and resampling to PC-MRA coordinate space in accordance with the resolution of PC-MRA.Registration of CE-MRA segmentation to 4D-flow was assessed by Dice similarity coefficient (DSC)  and averMean velocity and stasis were calculated to compare the functional properties of LA from the two segmentation methods. For stasis, the velocity threshold was set to 0.1 m/s based on previous studies .2.7.Statistics were analyzed using IBM SPSS Statistics for Windows, version 29.0 . Shapiro-Wilk test of normality was conducted then variables were reported as mean ± standard deviation (SD) if normally distributed or median [interquartile range]. For volume comparisons, a two-tailed paired t-test was used to evaluate differences. Pearson's correlation method was used to determine the correlation between two variables. Bland–Altman analysis was reported with the mean differences and 95% confidence intervals for the limits of agreement.3.3.1.2DS2-VASc score was 1.0. Detailed risk factors are described in Demographics of LA volume segmentation subjects are summarized in 3.2.p < 0.001). The volume ratio of the two segmentations was 1.24 ± 0.26, and the two volumes have a strong positive association .The mean of DSC of the registered cases was 0.770 ± 0.045, and the mean of ASSD was 2.704 mm ± 0.668 mm . Shapiro3.4.p = 0.286) in 41 successful registration cases. Individual data points were strongly correlated . Nevertheless, the two datasets were strongly correlated , and there was no proportional bias  CPU with 32 GB of RAM. Although the segmentation time was largely dependent on data quality and user experience, the semi-automated segmentation method with CE-MRA reduced user interaction compared to manual segmentation from scratch. This method currently requires additional time to convert file formats and switch operating platforms. However, this would eventually be reduced or eliminated by software integration. This study focused on LA segmentation, so the roughly outlined LAA was separated from the LA only to obtain a consistent LA segmentation. However, the measured size of the LAA was in agreement with previous studies. Moreover, we were able to clearly observe the shape and border of the LAA with CE-MRA, thanks to the approximately 2.5–3 times higher spatial resolution than with 4D-flow. If we make sufficient efforts to precisely define the LAA, this method will be advantageous for the 4D-flow study of the LAA.5.This method required manual input in several steps, so the result may still depend on the operator. The seed-and-grow method required not only seed input but also manual refinement afterwards, and the registration success or failure was determined by visual inspection. An automatic method to judge the registration results based on the flow pattern within the segmentation could be investigated in the future.All cases were acquired from the database of patients with paroxysmal AF patients prior to ablation. While their 4D-flow was acquired in sinus rhythm, AF induces structural alteration, so they should have had some degree of dilation. Application of this method to healthy controls and persistent AF patients will assess the generalizability of this method.For the excluded cases prior to volume segmentation, we do not know all the reasons for the low quality, but in most cases it may be an arrhythmia. Although patients were supposed to be in sinus rhythm, it is not uncommon for them to have AF episodes during the 4D-flow scan. Since 4D-flow requires in-line image reconstruction following a long acquisition, it is difficult to detect acquisition and reconstruction errors and rescan the patient during busy clinical routine examinations. Several of the excluded cases presented stripes generated during the reconstruction process. These stripes cannot be corrected during pre-processing. A possible solution is to repeat the in-line reconstruction in the scanner using the acquired raw data. However, this task may be difficult to perform in high-demand clinical routine examinations given the limited time during the scan sessions and the qualifications required to perform this advanced task in the scanner.In about a quarter of cases, registration failed even after the second attempt. Precise setting of ROI in both CE-MRA and PC-MRA segmentation, for example, contouring along the entire heart, may improve registration. However, it will add another operator manipulation and processing time; therefore, it was not considered. In this study, we only used time-averaged PC-MRA for signal-to-noise ratio, but this method smooths the moving structure to make a difference with the CE-MRA image. Registration with PC-MRA of the individual time phase or the average of a short period may be applied in the future.Our methodology showed a DSC of 0.77 and a ASSD of 2.7 mm which indicates noticeable differences between the manual LA segmentation from the PC-MRA and the LA segmentation from the CE-MRA. The resampling process into a lower resolution may increase the matching error between volumes. This is an important limitation that needs to be addressed in future work of the study.Flow was evaluated using mean velocity and stasis. Several other metrics, such as peak velocity, kinetic energy and vortex, have been used in previous studies . Based oFurthermore, this study did not included inter- and intra-variability analysis and scan-rescan assessment. Unveiling the variability and repeatability of the proposed approach must be considered in the continuity of this study.6.The proposed CE-MRA segmentation and registration method can generate segmentation for 4D-flow MRI images. The transformed LA segmentation moderately matches the 4D-flow image and produces analysis results compatible with the conventional segmentation method. The use of this method will facilitate 4D-flow analysis for AF patients by making segmentation easier and overcoming the limitation of resolution and contrast.


There is an urgent need to realize precise clinical ultrasound with ultrasound contrast agents that provide high echo intensity and mechanical index tolerance. Graphene derivatives possess exceptional characteristics, exhibiting great potential in fabricating ideal ultrasound contrast agents. Herein, we reported a facile and green approach to synthesizing reduced graphene oxide with ellagic acid (rGO-EA). To investigate the application of a graphene derivative in ultrasound contrast agents, rGO-EA was dispersed in saline solution and mixed with SonoVue (SV) to fabricate SV@rGO-EA microbubbles. To determine the properties of the product, analyses were performed, including ultraviolet–visible spectroscopy (UV–vis), Fourier-transform infrared spectroscopy (FTIR), Raman spectroscopy, transmission electron microscopy (TEM), thermal gravimetric analysis (TGA), X-ray photoelectron spectrum (XPS), X-ray diffraction analysis (XRD) and zeta potential analysis. Additionally, cell viability measurements and a hemolysis assay were conducted for a biosafety evaluation. SV@rGO-EA microbubbles were scanned at various mechanical index values to obtain the B-mode and contrast-enhanced ultrasound (CEUS) mode images in vitro. SV@rGO-EA microbubbles were administered to SD rats, and their livers and kidneys were imaged in CEUS and B-mode. The absorption of rGO-EA resulted in an enhanced echo intensity and mechanical index tolerance of SV@rGO-EA, surpassing the performance of SV microbubbles both in vitro and in vivo. This work exhibited the application potential of graphene derivatives in the field of ultrasound precision medicine. The past decades have witnessed enormous advancements in graphene research since the initial isolation of this two-dimensional miracle material. As an allotrope of carbon containing sp2-bonded atoms, graphene has impressive features, such as exceptional mechanical, electrical, optical and thermal characteristics. The ever-increasing interest in graphene-based materials has been focused on biomedical science in recent years. Additionally, inspirational research on graphene derivatives has been focused on wearable electronics, diagnostic tools, drug-delivery carriers and regenerative medicine materials ,2,3,4. HIn the ultrasound medical area, contrast-enhanced ultrasound (CEUS) is an important imaging technique, specialized for acquiring sensitive blood flow and tissue perfusion information. Ultrasound contrast agents (UCAs), the coated microbubbles that are stable in vivo, can be used to induce cavitation for diagnostic and therapeutic applications with suitably moderate ultrasound energy. This contributes to the development of accurate and subtle CEUS imaging. For decades, further contributions to enhanced cavitation effects and therapeutic delivery have witnessed the significance of UCA in molecular imaging and precise treatment ,6. The aSonoVue (SV) is a UCA approved by the Food and Drug Administration for liver ultrasound (US) imaging . SV micrGreen reducing agents, such as edible sunflower oil, lemon grass extracts and green tea extracts, can be employed for the eco-friendly synthesis of rGO ,13,14. EIn this work, we have explored the utilization of ellagic acid to develop a one-step, facile and eco-friendly approach for the preparation of rGO, avoiding expensive equipment, the need for a rigorous environment and the presence of toxic constituents inherent in conventional chemical synthesis . Carbon As a natural polyphenol, ellagic acid exhibits reduction properties. The GO solution with EA underwent sonication and exhibited an initial brown coloration. Upon completion of the reaction, the solution turned a deep, dark hue. −1, corresponding to the C=O stretching of carboxylic and/or carbonyl moiety functional groups, along with a peak at 1202 cm−1 associated with C–O stretching vibrations. Additionally, the presence of unoxidized graphitic domains was evidenced by the peak observed at 1555 cm−1 due to the skeletal vibrations [−1, which were associated with the characteristic peaks of EA, suggesting that EA was successfully conjugated [−1 are presented in −1 and the D band at 1348 cm−1, indicating the presence of amorphous carbon [1g mode of k-point photons, and the G band in E2g mode was associated with the ordered sp2-bonded carbon atoms in rGO [−1, corresponding to the D and G bands. The calculated ID/IG value for rGO-EA was 0.96, surpassing that of GO, indicating the functionalization of rGO [A series of characterizations was conducted to confirm the properties of the product. The reduction was monitored by measuring the position of the UV–vis absorption peak a. The UVbrations ,26. The s carbon . The D bs in rGO . The intn of rGO . As GO un of rGO . A new pn of rGO . This ren of rGO . In addin of rGO .The TGA method demonstrated high precision in examining the composition and thermal stability changes in composites. The TGA thermograms illustrating the characteristics of GO, EA and rGO-EA are presented in An XPS analysis was carried out to analyze the impact on the carbon and oxygen contents of the reduction process. TEM examinations were carried out to describe the morphological characteristics of GO and rGO-EA. The SAED patterns of GO and rGO-EA are also shown in The in vitro cell viability of rGO-EA on the 4T1 breast cancer cell line and the NIH3T3 fibroblast cell line were obtained using CCK8 analysis to evaluate the metabolic activity of cultured cells. IBM SPSS Statistics 22 software was utilized to determine the half-maximal inhibitory concentration (IC50) of rGO-EA against 4T1 cells. As shown in Hemolysis (%) = (ODe − ODn)/(ODp − ODn) × 100%, where ODe, ODn and ODp correspond to the absorbance values of the experimental, negative and positive groups, respectively. Overall, hemolysis increased with the rising SV@rGO-EA concentration (up to 4.1% at 0.2 mg/mL), but this was insignificant (<5%).Hemolysis is attributed to the damage of the RBC membrane, leading to hemoglobin being released into the surrounding solution. Generally, a hemolysis level less than 5% indicates a feasible biomaterial . Figure The TEM image reveals the spherical morphology of the SV MBs in The CEUS and B-mode ultrasound images of SV and SV@rGO-EA are presented in The ultrasound enhancement assessment of SV and SV@rGO-EA at 0.08 MI in the animal body was carried out using SV as the control group, and B-mode imaging was recorded at the same time in comparison with CEUS. We took the results of the biosafety assay and ultrasound imaging in vitro into consideration, and the rGO-EA concentration in SV@rGO-EA MBs was set at 0.10 mg/mL, which provided impressive echo intensity and CEUS imaging in vitro. The equivalent SV concentration was 5 mg/mL in every group. The CEUS and B-mode imaging effect on rat livers and kidneys is presented in A quantitative analysis of the mean echo intensity of livers and kidneys in the drawn ROIs in To sum up, rGO-EA nanosheets with a monolayer gauzy structure b could i® . SonoVue (sulfur hexafluoride microbubbles for injection) was purchased from Bracco Sine Pharmaceutical Co., Ltd. .Graphene oxide (GO) was purchased from Sailing Commercial Co., Ltd. . Ellagic acid (CAS: 476-66-4) was purchased from Shanghai Macklin Biochemical Technology Co., Ltd. . Tris(hydroxymethyl)aminomethane hydrochloride (CAS: 1185-53-1) was purchased from Shanghai Aladdin Bio-Chem Technology Co., Ltd. . RPMI 1640 medium (PM150110A) and Dulbecco’s Modified Eagle’s Medium  were purchased from Procell Life Science & Technology Co., Ltd. . Phosphate-buffered saline (PBS) was purchased from Biosharp−1 penicillin/streptomycin. The NIH3T3 cell line was cultured in DMEM medium with 10% fetal bovine serum and 100 IU mL−1 penicillin/streptomycin. Female SD rats (6–8 weeks old) with body weights of 150 g were obtained from the Laboratory Animal Center of Zhengzhou University (China).The murine 4T1 cell line (CL-0007) and NIH3T3 cell line (CL-0171) were kindly provided by Procell Life Science & Technology Co., Ltd. (China). The 4T1 cell line was cultured in an RPMI 1640 medium with 10% fetal bovine serum and 100 IU mLFor the green synthesis of rGO-EA, 400 mg of EA powder and 100 mg of GO were added to a beaker containing 100 mL distilled water. An appropriate dosage of Tris was mixed into the solution for even distribution, and the pH was adjusted to 8.5. After ultrasonication for 2 h, the well-dispersed solution was stirred in the condition of 70 °C for 24 h. The final product was diluted and washed with deionized water three times to ensure the complete removal of residual EA and Tris. It was freeze-dried for further use.To determine the characteristics of GO and rGO-EA, a Fourier-transform infrared (FTIR) spectrophotometer , ultraviolet–visible (UV–vis) spectrophotometer , X-ray diffraction (XRD) instrument , X-ray photoelectron spectrum (XPS) instrument  and a thermal gravimetric analysis (TGA) measurement  were utilized. Furthermore, a transmission electron microscopy (TEM) microscope  was used to confirm the morphology. The zeta potential of the GO and rGO-EA samples was determined five times at 25 °C using a potentiometric analysis instrument . Briefly, 5 mL sterile normal saline was added to the SonoVue vial using a syringe, then shaken vigorously for 20 s to mix the contents to obtain SV MBs (5 mg/mL). A SonoVue vial was filled with white lipid powder and sulfur hexafluoride gas. Sterile normal saline was shaken well in the vial to obtain a white milky homogeneous liquid, which contained sufficient SV MBs. SV@rGO-EA MBs were produced after the mixture of rGO-EA saline solution and the homogeneous liquid with SV MBs, which showed a light gray hue. To be specific, 2mg of rGO-EA was added to 5 mL saline and sonicated for 1 h to achieve uniform dispersion. The rGO-EA solution (0.40 mg/mL) was mixed with an equal volume of SV (5mg/mL) to create SV@rGO-EA MBs, with an equivalent concentration of rGO-EA at 0.20 mg/mL. Similarly, vials containing self-assembly SV@rGO-EA MBs were dispersed in saline solution, resulting in equivalent concentrations of rGO-EA at 0.05 and 0.10 mg/mL.The zeta potential of SV and SV@rGO-EA MBs solutions was analyzed on a Zetasizer Nano ZS90 analyzer . The measurement was repeated at least five times, and average values of zeta potential were adopted. A TEM microscope  was also employed to observe the structural and morphological characterizations of SV and SV@rGO-EA MBs.2. Subsequently, wells were treated with 200 μL of culture medium containing different concentrations of rGO-EA for an additional 24 h under the same environment. The control wells were simply filled with culture medium and cells. Following three washes of the culture with 200 μL of PBS solution, each well was supplemented with 10 μL of CCK-8 assay and 100 μL of culture medium under identical conditions for 2 h. Cell viability was assessed using a microplate reader  by measuring absorbance at 450 nm and calculated as a percentage relative to the control (cells grown without the samples were considered to have 100% viability).The growth-inhibiting ability of rGO-EA was preliminarily evaluated using the Cell Counting Kit-8 (CCK-8) assay. The 4T1 and NIH3T3 cells were respectively seeded in 96-well assay plates for 24 h under conditions of 37 °C in a humidified incubator with 5% COA hemolysis assay was performed to further evaluate the toxicity of the final products. A solution of SV@rGO-EA was prepared with sterile normal saline and kept at 37 °C for 30 min. An experimental group with a 4% RBC suspension (0.5 mL) was added to the SV@rGO-EA suspensions with a concentration gradient (0.5 mL) at 37 °C for 4h. Deionized water or sterile normal saline (0.5 mL) was added to 0.5 mL of 4% RBC suspension as a positive and negative control, respectively. The mixed suspensions were centrifuged at 3000 rpm for 5 min to collect the supernatant. The absorbance values of the supernatants were measured at 545 nm with a microplate reader  to detect the released hemoglobin from the RBCs.The plastic pipettes were separated into three groups, filled with each concentration of SV@rGO-EA MBs solution and securely sealed by clamps. The concentrations of SV@rGO-EA solutions were 0.05, 0.10 and 0.20 mg/mL, respectively. The pipette filled with SV MBs served as a control. A large beaker, filled with a thick sound-absorbing sponge at the bottom, was filled with degassed distilled water. The pipettes containing samples were then immersed into the beaker at 25 °C. The ultrasound transducer was maintained parallel to the long axis of the pipettes, which were submerged in water. All the samples underwent scanning using the Hivision Ascendus ultrasound diagnostic system  in both B-mode and CEUS mode. The MI values were set at 0.10, 0.20, 0.40, 0.60, 0.80 and 1.00, respectively. The focus was adjusted within the range of depths from 1.2 cm to 2.0 cm, while maintaining an imaging target depth of 4 cm. The real-time observation of ultrasound contrast-enhanced imaging was conducted, and the videos were stored.SD rats were randomly divided into two groups with four rats in each. After the general anesthesia via an intraperitoneal injection of 10 mg/mL pentobarbital sodium (40 mL/kg), which could be sustained for 2 h, the rats were placed on their backs on heated blankets with their limbs fastened appropriately. The white fur on the chest and belly was depilated.The in vivo imaging experiments were conducted using a PHILIPS EPIQ 7 ultrasound diagnostic system  equipped with a L12-5 linear array transducer (5–12 MHz). In response to the results of ultrasound imaging in vitro, a MI of 0.08 was selected for the contrast-enhanced imaging of MBs in SD rat livers and kidneys. Prior to contrast agent injection, CEUS and B-mode imaging were performed on rats’ livers and kidneys. Then, the control was administered with 200 μL of intravenously injected SV, and the other group received an intravenous injection with 200 μL SV@rGO-EA MBs . At the conclusion of the experiments, euthanasia was performed on the SD rats.SV with a delicate lipid shell will be significantly disrupted at high MIs. Fortunately, ultrasonic irradiation with a MI of 0.08 can generate an ideal US echo intensity for a considerable duration. To elucidate the alterations in echo intensity, the livers and kidneys were imaged within 50 s following intravenous injection. The echo intensities of the livers and kidneys in rats imaged with each UCA (SV@rGO-EA and SV) were quantitatively analyzed using QLAB10, a specialized quantitative analytical software, in conjunction with the diagnostic color Doppler ultrasound system. The intensity values within specific regions of interest (ROIs) were also subjected to further analysis.t test was used for comparisons between two groups. Data among three or more groups were analyzed according to one-way ANOVA followed by a Bonferroni post hoc test. The significance level was considered p < 0.05.All data were analyzed using IBM SPSS Statistics 22 software. Measurement data were presented as mean ± standard deviation. Student’s In this work, GO was successfully reduced by ellagic acid, with EA loaded on the surface of rGO. The convenient one-step green reduction of graphene oxide and the adsorption of EA were clarified by various characterization techniques, including UV–vis, FTIR, Raman, XRD, TGA, XPS, TEM and zeta potential analysis. Additionally, the absorption of rGO-EA onto the spherical surface of SV MBs was successfully achieved. The biosafety of the product was confirmed through a cell viability assay and hemolysis assay. The results produced a good biomedical material, enhancing echo intensity and MI tolerance and surpassing the performance of SV MBs both in vitro and in vivo. The application of graphene derivatives in UCAs has been preliminarily investigated, with various graphene derivatives being considered as promising materials for extensive research and development in the medical ultrasound field.